One of the techniques for assaying for antibodies to microbial organisms involves forming a conjugate between the antibody and an antigen derived from the microbe such as, for example, from the cell wall thereof. The conjugate is precipitated from the test solution by the presence of an anti-antibody derived from a different mammalian species. By introducing a suitable label into the conjugate utilizing either a labelled antigen or a labelled anti-antibody, it is possible to determine the concentration of the antibody in the sample using a previously generated standard curve.
Thus, for example, detection of gonorrhea antibodies in human serum is described in U.S. Pat. No. 3,974,269. The method is based on the use of an antigen produced by the Neisseria gonorrhoeae organism whose isolation is described in further detail in U.S. Patent Application Ser. No. 385,863 filed Aug. 6, 1973.
In a specific embodiment of a radioimmunoassay described in U.S. Pat. No. 3,974,269, N.g. antibodies in serum are detected by a process comprising:
A. Adding anti-human IgG to the serum to be tested in a buffer aqueous medium;
B. Thereafter adding a heat labile antigen labelled with a radioactive element;
C. Incubating the resulting mixture at from about 4.degree. to 45.degree. C. for from about 24 to 2 hours at a pH of from about 6.5 to 8.5 to form an antigen-antibody conjugate when antibodies are present in the serum; and
D. Determining the level of radioactivity as a measure of the presence of the antigen-antibody conjugate.
The precipitated conjugate is separated from the solution by either filtration or centrifugation. In accordance with the prior knowledge and practice in the art, the amount of anti-human IgG used in relation to the amount of serum in step A is adjusted to maximize precipitation. The assumption has always been that maximum precipitation is equivalent to maximum radioactivity yield. To this end the ratio of anti-human IgG antibody to serum sample volumes is adjusted to substantially more than 1/1, usually a ratio of 6/1 is used.
Several problems remain before such techniques can be utilized as the basis for mass screening tests. One major problem is the fact that the positive cut off level is three standard deviations above the mean of a group of negative sera even at maximum sensitivity. Thus, the test would pick up positive sera having relatively high antibody titers but would not accurately pick up sera having weak or borderline concentrations of antibody which still represent subjects with positive infections of the microbe agents in question. Moreover, in order to reach this level of sensitivity it is critically necessary to add the reagents in specific order, that is the anti-human IgG is added to the buffered test serum followed by addition of the labelled antigen. Use of the normal order wherein the antigen is added to the serum followed by the anti-human IgG results in a test which is indicated to be of insufficient sensitivity to distinguish between positive and negative sera with a useful degree of confidence.
A solid-phase radioimmunoassay for rubella virus is described by Kalimo et al., J. Clin. Microbiol. 4, No. 2, 117 (1976). This assay employs rubella virus adsorbed onto polystyrene balls.